B2S Life Sciences LLC, 97 East Monroe Street, Franklin, IN, 46131, USA.
Sci Rep. 2021 Jul 29;11(1):15467. doi: 10.1038/s41598-021-95055-x.
Today the evaluation of unwanted immunogenicity is a key component in the clinical safety evaluation of new biotherapeutic drugs and macromolecular delivery strategies. However, the evolving structural complexity in contemporary biotherapeutics creates a need for on-going innovation in assay designs for reliable detection of anti-drug antibodies, especially for biotherapeutics that may not be well-suited for testing by a bridging assay. We, therefore, initiated systematic optimization of the direct binding assay to adapt it for routine use in regulatory-compliant assays of serum anti-drug antibodies. Accordingly, we first prepared a SULFO-TAG labeled conjugate of recombinant Protein-A/G to create a sensitive electrochemiluminescent secondary detection reagent with broad reactivity to antibodies across many species. Secondly, we evaluated candidate blocker-diluents to identify ones producing the highest signal-to-noise response ratios. Lastly, we introduced use of the ratio of signal responses in biotherapeutic-coated and uncoated wells as a data transformation strategy to identify biological outliers. This alternative data normalization approach improved normality, reduced skewness, and facilitated application of a parametric screening cut point. We believe the optimized direct binding assay design employing SULFO-TAG labeled Protein-A/G represents a useful analytical design for detecting serum ADA to biotherapeutics that lack an immunoglobulin Fc domain.
今天，评估不必要的免疫原性是新生物治疗药物和大分子输送策略临床安全性评估的关键组成部分。然而，当代生物治疗学中不断发展的结构复杂性要求不断创新检测抗药物抗体的检测方法设计，特别是对于那些可能不适合桥接检测的生物治疗药物。因此，我们开始对直接结合检测进行系统优化，以适应常规使用，对血清抗药物抗体进行监管合规检测。因此，我们首先制备了 SULFO-TAG 标记的重组蛋白 A/G 缀合物，以创建一种对许多物种的抗体具有广泛反应性的灵敏电化学发光二级检测试剂。其次，我们评估了候选阻滞剂稀释剂，以确定产生最高信噪比的那些。最后，我们引入了生物治疗药物包被和未包被孔中信号响应比值的使用，作为数据转换策略来识别生物学异常值。这种替代数据归一化方法提高了正态性，降低了偏度，并有助于应用参数筛选切点。我们相信，采用 SULFO-TAG 标记的蛋白 A/G 的优化直接结合检测设计代表了一种用于检测缺乏免疫球蛋白 Fc 结构域的生物治疗药物的血清 ADA 的有用分析设计。
